Plant disease outbreaks have resulted in catastrophic crop failures that have triggered famines and caused major social change. Generally, the best strategy for plant disease control is to use resistant cultivars selected or developed by plant breeders for this purpose. However, the potential for serious crop disease epidemics persists today, as evidenced by outbreaks of the Victoria blight of oats and southern corn leaf blight. An additional intractable problem facing farmers worldwide is the contamination of crops by mycotoxins, particularly aflatoxin (AF), a potent carcinogen. During years of high temperatures and drought stress, invasion of kernels by opportunistic seed infesting fungi of the Aspergillus genus, namely A. parasiticus and A. flavus, is prevalent. These fungi readily produce AFB1, leading to substantial crop loss in developed countries, and significant AF associated health problems in less wealthy countries unable to implement detection and decontamination strategies. Because traditional plant protection and breeding methods are not sufficient to prevent this disease, research efforts have turned to deciphering the molecular events regulating the Aspergillus/seed interaction as a means to develop effective control measures. Accordingly, molecular methods are needed to supplement traditional breeding methods to protect plants from pathogen attack and to reduce levels of mycotoxin.
Lipoxygenase (LOX) is a nonheme iron-containing dioxygenase that catalyzes the regio- and stereo-selective dioxygenation of polyunsaturated fatty acids forming hydroperoxy derivatives. Plant lipoxygenases are classified into 9- and 13-LOXs with respect to their positional specificity of linoleic acid (LA) oxygenation. Widely distributed in the plant and animal kingdom, lipoxygenases play a number of roles. In mammalian cells, lipoxygenases are involved in the biosynthesis of molecules, which mediate inflammatory responses in different tissues. In plants, lipoxygenase expression has been correlated with growth and development, maturation, senescence, wounding and stress, pathogen attack, and biosynthesis of signaling molecules such as jasmonic acid and methyl jasmonate. Increases in host lipoxygenase activity and individual lipoxygenase isozymes after infection with bacterial and fungal pathogens have been observed in a number of plant species.
Oxidation of unsaturated C18 fatty acids by lipoxygenases results in the formation of 9-hydroperoxy 10(E), 12(Z)- and 13-hydroperoxy-9(Z), 11(E)-derivatives of linole(n)ic acids. The hydroperoxy devivatives can serve as substrates for further transformation by 1) the peroxygenase pathway producing epoxides, epoxy alcohols, dihydrodiols/triols; 2) the hydroperoxide lyase pathway producing aldehydes, oxo-acids, and other C6 volatiles; and 3) with the 13-hydroperoxy derivatives, the allene oxide synthase pathway producing cyclized products, alpha- and gamma-ketols.
The present invention describes a maize lipoxygenase, CSSAP92, involved in not only disease resistance to pathogen but also in resistance to aflatoxin contamination. By modulating the level of CSSAP92 in a transgenic plant, it is possible to affect a plant""s resistance to disease and/or to reduce the level of aflatoxin contamination in grain.
Generally, it is the object of the present invention to provide nucleic acids and proteins relating to a maize lipoxygenase, CSSAP92. It is an object of the present invention to provide transgenic plants comprising the nucleic acids of the present invention. It is another object of the present invention to provide methods for modulating, in a transgenic plant, the expression of the nucleic acids of the present invention.
Therefore, in one aspect, the present invention relates to an isolated nucleic acid comprising a member selected from the group consisting of (a) a polynucleotide encoding a polypeptide of the present invention as shown in SEQ ID NO: 3; (b) the polynucleotide having the sequence found in SEQ ID NO: 1 or SEQ ID NO: 2 and (c) a polynucleotide complementary to a polynucleotide of (a) and (b). The isolated nucleic acid can be DNA. The isolated nucleic acid can also be RNA.
In another aspect, the present invention relates to vectors comprising the polynucleotides of the present invention. Also the present invention relates to recombinant expression cassettes, comprising a nucleic acid of the present invention operably linked to a promoter.
In another aspect, the present invention is directed to a host cell into which has been introduced the recombinant expression cassette.
In yet another aspect, the present invention relates to a transgenic plant or plant cell comprising a recombinant expression cassette with a promoter operably linked to any of the isolated nucleic acids of the present invention. Preferred plants containing the recombinant expression cassette of the present invention include but are not limited to maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice barley, and millet. The present invention also provides transgenic seed from the transgenic plant.
In another aspect, the present invention relates to a polypeptide characterized by SEQ ID NO: 3.
In further aspect, the present invention relates to a method of modulating the level of protein in a plant by introducing into a plant cell a recombinant expression cassette comprising a polynucleotide of the present invention operably linked to a promoter; culturing the plant cell under plant growing conditions to produce a regenerated plant; and inducing expression of the polynucleotide for a time sufficient to modulate the protein of the present invention in the plant.
In a further aspect, the present invention relates to a method of decreasing aflatoxin contamination in a plant by introducing into a plant cell a recombinant expression cassette comprising a polynucleotide of the present invention operably linked to a promoter; culturing the plant cell under plant growing conditions to produce a regenerated plant; and inducing expression of the polynucleotide for a time sufficient to reduce aflatoxin contamination in the plant. The polynucleotide may be in the sense or antisense orientation.
In addition, the present invention relates to a method of finding maize lines with reduced levels of CSSAP92 protein comprising the steps of a) generating antibodies to the polypeptide of SEQ ID NO: 3; b) screening maize seeds and identifying seeds with low levels of the polypeptide; c) identifying and cloning the corresponding polynucleotide of the polypeptide; d) identify the allele-specific variations of the cloned polynucleotide; and e) screen maize lines for the allele-specific variations to determine plant lines containing the allele-specific variations.
In another aspect, the present invention relates to a method of increasing 13S-HPODE production in a plant comprising the steps of cloning a polynucleotide which encodes for a N-terminally truncated polypeptide of the present invention operably linked to a promoter, introducing into a plant cell the expression cassette, regenerating a plant from the transformed plant cell, and inducing expression of the fragment for a time sufficient to increase 13S-HPODE production. Preferably the fragment is the 2.6 Kb SalI-NotI fragment of SEQ ID NO: 1.
Further, the present invention relates to methods of increasing resistance in a plant to a pathogen comprising introducing into a plant cell a recombinant expression cassette comprising a polynucleotide of the present invention operably linked to a promoter, regenerating a plant from the transformed plant cell, and inducing expression of the polynucleotide for a time sufficient to increase resistance to a pathogen in the plant. The promoter may be a pathogen-inducible promoter or a tissue-preferred promoter.
Preferred plants and plant cells of the present invention include but are not limited to maize, soybean, sunflower, sorghum, canola, wheat, alfalfa, cotton, rice, barley, and millet. The level of protein in the plant can either be increased or decreased. The polynucleotide can be either in the sense or antisense direction.